Morphological and phylogenetic evidence reveal three new Pseudohydnum (Auriculariales, Basidiomycota) species from North China

Pseudohydnum is characterized by gelatinous basidiomata with hydnoid hymenophores and longitudinally septate basidia. In this study, samples of the genus from North China were examined morphologically and phylogenetically using a dataset of the internal transcribed spacer of the ribosomal RNA gene and the nuclear large subunit rDNA. This study describes three new species, namely Pseudohydnum abietinum, Pseudohydnum candidissimum, and Pseudohydnum sinobisporum. Pseudohydnum abietinum is characterized by pileate and pale clay pink basidiomata when fresh, with a rudimentary stipe base, four-celled basidia, and broadly ellipsoid to ovoid or subglobose basidiospores (6−7.5 × 5−6.3 μm). P. candidissimum is characterized by very white basidiomata when fresh, frequently four-celled basidia, and broadly ellipsoid to subglobose basidiospores (7.2−8.5 × 6−7 μm). P. sinobisporum is characterized by ivory basidiomata when fresh, two-celled basidia, ovoid to broadly ellipsoid, or subglobose basidiospores (7.5−9.5 × 5.8−7.2 μm). The main characteristics, type localities, and hosts of Pseudohydnum species are listed.

. However, Ingold (1982;1985) noted that Pseudohydnum and Exidia have a relatively close relationship based on spore germination. Morphologically, Bandoni (1984) redefined the concept of Auriculariales, and the family Aporpiaceae was used to accommodate taxa with myxarioid basidia, including Pseudohydnum. Weiss and Oberwinkler (2001) verified that Pseudohydnum has a close relationship with Auriculariales based on phylogenetic analyses; however, the position of Pseudohydnum in Auriculariales was ambiguous.
During an investigation of jelly fungi in North China, several samples belonging to Pseudohydnum were collected, and three unknown species were found. To confirm the affinity of the taxa, phylogenetic analysis was performed based on the internal transcribed spacer (ITS) and large subunit nuclear ribosomal RNA gene (LSU) sequences.

Morphological studies
The specimens were collected from the provinces of Jinlin, Heilongjiang, and Gansu in North China. They were deposited in the herbaria of Beijing Forestry University (BJFC) and the Mycology Department of Jinlin Agriculture University (HMJAU). Samples were photographed when fresh in the field, and their habitats were recorded. Microscopic structures were discussed by Chen et al. (2020), Fan et al. (2021), andZhou et al. (2022). Special color terms were set by Anonymous (1969) and Petersen (1996). A Nikon Digital Sight DS-L3 or Leica ICC50 HD camera (magnification ×1,000) was used to examine hand-cut sections of basidiomata, which were first treated with 5% KOH for a few minutes and then with 1% phloxine B (C 20 H 4 Br 4 Cl 2 K 2 O 5 ). At least 30 basidiospores of each species were examined. The values were expressed as a mean with 5% of the measurements excluded from each end of the range, given in parentheses. Stalks were excluded for basidia measurement, and the hilar appendages were excluded for basidiospore measurement.
The following abbreviations are used in the descriptions: IKI, Melzer's reagent; IKI−, neither amyloid nor dextrinoid; CB, cotton blue; CB−, acyanophilous in cotton blue; L, the arithmetic average of spore lengths; W, the arithmetic average of spore widths; Q, L/W ratio; and n (a/b), number of spores (a) measured from a given number (b) of specimens.

DNA extraction, amplification, and sequencing
The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing) was used to obtain DNA from dried specimens and PCR was performed according to the manufacturer's instructions with some modifications (Chen and Dai, 2021). Two DNA gene fragments, ITS and LSU, were amplified using the primer pairs ITS5/ITS4 (White et al., 1990) and LR0R/LR7, respectively (Hopple and Vilgalys, 1994).
The PCR procedure for ITS was as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles at 94°C for 40 s, 54°C for 45 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR procedure for LSU was as follows: initial denaturation at 94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 50°C for 1 min, and 72°C for 1.5 min; and a final extension at 72°C for 10 min. DNA sequencing was performed at the Beijing Genomics Institute. All newly generated sequences were submitted to GenBank and are listed in Table 1.
Sequences generated for this study were aligned, with additional sequences downloaded from GenBank. Both ITS and LSU sequences were aligned using MAFFT v.7 (https://mafft.cbrc.jp/alignment/ server/), adjusting the direction of nucleotide sequences according to the first sequence (accurate enough for most cases), and selecting the G-INS-i iterative refinement method (Katoh et al., 2019). Alignments were manually adjusted to maximize alignment and minimize gaps with BioEdit v.7.0.9 (Hall, 1999). A dataset composed of concatenated ITS + LSU sequences was used to determine the phylogenetic position of new species. The aligned sequences were deposited in TreeBase (https://www.treebase.org/ treebase-web/home.html; submission ID 29962). Protomerulius subreflexus (Lloyd) O. Miettinen & Ryvarden and P. substuppeus (Berk. & Cooke) Ryvarden were selected as outgroups following Chen et al. (2020).
Maximum likelihood (ML) analysis was performed using the CIPRES Science Gateway (Miller et al., 2009) based on the dataset using the RA × ML-HPC BlackBox tool, with setting RA × ML halt bootstrapping automatically and 0.25 for maximum hours and obtaining the best tree using ML search. Other parameters in ML analysis used default settings, and statistical support values were obtained using nonparametric bootstrapping with 1,000 replicates.
Bayesian inference (BI) analysis based on the dataset was performed using MrBayes v.3.2.6 (Ronquist et al., 2012). The best substitution model for the dataset was selected by ModelFinder (Kalyaanamoorthy et al., 2017) using a Bayesian information criterion, and the model was used for Bayesian analysis. Four Markov chains were run from random starting trees for 0.8 million generations. Trees were sampled every 1,000th generation. The first 25% of sampled trees were discarded as burn-in, whereas other trees were used to construct a 50% majority consensus tree and for calculating Bayesian posterior probabilities (BPPs).

Phylogeny
The concatenated ITS + LSU dataset included 30 ITS and 22 LSU sequences from 30 samples representing 14 taxa. The best model for the concatenated ITS + LSU dataset estimated and applied for BI analysis was "SYM + I + G", datatype = DNA, nucmodel = 4by4, lset nst = 6, rates = invgamma; state frequencies had a Dirichlet prior (1,1,1,1), and the distribution was approximated using four categories. BI analysis yielded a similar topology to ML analysis, with an average standard deviation of split frequencies of 0.007485. The ML tree was provided ( Figure 1). Branches that received bootstrap support for ML (ML-BS) and BI (BPP) ≥70% (ML-BS), and 0.85 (BPP) were considered significantly supported, respectively.
Phylogenetic analysis placed all Pseudohydnum samples in a fully supported clade (100/1, Figure 1). Five specimens from Northeast China formed two lineages, namely P. candidissmum and P. sinobisporum, clustered with P. himalayanum as strong support (100/1, Figure 1). The two specimens from Northwest China were named P. abietinum, sister to P. sinogelatinosum and P. gelatinosum. The samples from North America were treated as "P. gelatinosum-1" and "P. gelatinosum-2." Diagnosis-Differed from other Pseudohydnum species in having pileate basidiomata, with a rudimentary stipe base, pale clay pink pileal surface when fresh, hymenophore with spines 2−3 per mm at the base, broadly ellipsoid to ovoid or subglobose basidiospores measuring 6−7.5 × 5−6.3 mm, and occurring in Gansu Province, Northwest China.
Basidiomata-Annual, pileate with a rudimentary stipe base, gelatinous when fresh, brittle when dry, usually solitary. Pilei were dimidiated to flabelliform, projecting up to 1.5 cm, 1.4 cm wide, and 1.9 mm thick when dry. Pileal surfaces were pale clay pink when fresh, and hazel when dry. Spines were white and conical when fresh, cream when dry, 2−3 per mm at the base, and up to 1.5-mm long when dry. The context was translucent when fresh.
Etymology-Candidissimum (Lat.): referring to the species having very white basidiomata when fresh.
Basidiomata-Annual, gelatinous when fresh, brittle when dry, usually solitary, with a lateral stipe. Pilei flabelliform to dimidiate, projecting up to 1.5 cm, 1.2 cm wide, and 0.6-mm thick when dry. The pileal surface was white when fresh and pale mouse-gray when dry. Spines were white and conical when fresh, buff-yellow when dry, 2−3 per mm at the base, and up to 0.5-mm long. The context was translucent when fresh. Stipe concolorous with pileal surface, translucent when fresh, up to 5-mm long and 3 mm in diam. when dry.
Etymology-Sinobisporum (Lat.): referring to the species having two spores on each basidium and being found in China.
Basidiomata-Annual, gelatinous when fresh, brittle when dry, solitary, with a lateral stipe. Pilei was shell-shaped, projecting up to 1.2 cm, 1 cm wide, and 1.2 mm thick when dry. Pileal surfaces were ivory when fresh and hazel when dry. Spines were white and conical when fresh, cream when dry, 2−3 per mm at the base, and up to 1 mm long when dry. The context was translucent when fresh. Stipe concolorous with pileal surface, shrinking to the base, translucent when fresh, up to 5.5-mm long and 5 mm in diam. when dry.
In this study, three new species of Pseudohydnum were identified in North China: P. abietinum, P. candidissimum, and P. sinobisporum.
Jelly fungi are a special group of wood-inhabiting basidiomycetes and most species belong to the taxa form phragmobasidia (Wells, 1994). Most belong to Auriculariales and Tremellales, and some are edible mushrooms (Dai et al., 2010;Luo et al., 2022;Yao et al., 2022;Zhang et al., 2022). However, the diversity of the Chinese jelly fungi is not well-known, and recently, new species were described from China based on both morphology and phylogeny Fan et al., 2021;Zhou et al., 2022). Advanced techniques, including molecular phylogeny and omics, will aid in discovering new species of jelly fungi in the future.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/ genbank/, OP965345-OP965351 and https://www.ncbi.nlm.nih.gov/ genbank/, OP965365-OP965371.   Lloyd (1925) New species are in bold. The symbol "-" represents that there is no sequence. substantial, direct, and intellectual contribution to the work and approved it for publication.

Funding
The research was supported by the National Natural Science Foundation of China (Nos. 32070016 and 32270016).